We propose to study the interactions that occur during three crucial stages of the HIV viral life cycle between HIV-1 proteins and cell membranes. For the stage of HIV budding to form new virions, we seek to understand the lipid requirements for the Gag matrix (MA) protein to bind to the T-cell plasma membrane and multimerize. For the stage of transporting the transcriptional transactivator Tat through the T-cell nuclear membrane, it is important to understand why transport requires a sequence of eight positively charged residues. A third important problem for both viral maturation and infectivity stages is to determine how the cytoplasmic C-terminal tail (CTT) of the HIV gp41 protein affects the ectodomain envelope proteins (Env) through an, as yet unknown, allosteric transmembrane mechanism. A possible second function of the CTT is to cause T-cell death by pore formation in the T-cell plasma membrane. Progress on these problems would provide a fundamental scientific basis for pharmaceutical research designed to manipulate lipid composition to provide resistance to viral infection. State-of-the-art X-ray and neutron scattering methods will be applied. Innovative methods developed in our lab provide structural data from out-of-plane low angle x-ray scattering (LAXS) on fully hydrated oriented samples. When combined with our volumetric measurements and with small angle neutron scattering (SANS) data, we determine many membrane properties, including membrane thicknesses, molecular areas, bending stiffness, and locations of peptides in the membrane. Our data will be used 1) to test an MA binding hypothesis involving highly negatively charged PI(4,5)P2 lipid and the MA myristoyl switch, 2) to test an hypothesis that Tat penetrates rather deeply into the membrane en route to forming a translocation pore, and 3) to study how the essential LLP-2 and LLP-1 peptides in CTT associate with membranes in order to effect transmembrane communication and pore formation. A unifying theme underlying these three problems is the importance of positively charged amino acid residues interacting with biomembranes. All three problems will benefit from our recent method to determine the membrane order parameter from our WAXS scattering measurements. We also propose to use in-plane x-ray and neutron scattering from our oriented, fully hydrated samples to study multimerization of MA or MA fragments that occurs in virion budding, and pore formation caused by Tat and by LLP-1. Lipid compositions will be chosen to mimic the T-cell plasma or nuclear membranes, or the HIV virion membrane, and systematic variations from these will test hypotheses and determine specific lipid requirements, such as for PI(4,5)P2, cholesterol and negatively charged lipids. The broad, long-term scientific goal of the proposed studies is to determine the effective interactions of the many different lipids, cholesterol and proteins that make up complex biomembranes. PUBLIC HEALTH RELEVANCE: This research will provide a fundamental scientific basis for potential pharmaceutical research designed to correct the lipid composition of diseased cell membranes or to provide healthy cells with resistance to viral infection. Our main focus is to study how HIV basic peptides interact with HIV and T-cell membranes to affect replication and infectivity at various stages in the HIV life cycle, which may lead to a new class of drugs to fight AIDS.